EP4001303B1 - Verfahren zur herstellung eines langwirkenden wirkstoffkonjugats durch herstellung eines neuartigen zwischenproduktes - Google Patents

Verfahren zur herstellung eines langwirkenden wirkstoffkonjugats durch herstellung eines neuartigen zwischenproduktes Download PDF

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EP4001303B1
EP4001303B1 EP19937656.7A EP19937656A EP4001303B1 EP 4001303 B1 EP4001303 B1 EP 4001303B1 EP 19937656 A EP19937656 A EP 19937656A EP 4001303 B1 EP4001303 B1 EP 4001303B1
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region
immunoglobulin
formula
factor
physiologically active
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French (fr)
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EP4001303A4 (de
EP4001303A1 (de
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Cheongbyeol SHIN
Dooseo JANG
Ji Hye Moon
Dong Hyun Kim
Ji Eun Lee
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Hanmi Pharmaceutical Co Ltd
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Hanmi Pharmaceutical Co Ltd
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Priority to HUE19937656A priority patent/HUE073094T2/hu
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/321—Polymers modified by chemical after-treatment with inorganic compounds
    • C08G65/325—Polymers modified by chemical after-treatment with inorganic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to a novel intermediate for preparing a long-acting drug conjugate, a composition including the same, and a method for preparing the long-acting drug conjugate using the same.
  • Physiologically active polypeptides are easily denatured due to low stability, degraded by proteases in the blood, and easily removed by the kidneys or liver.
  • the protein drug needs to be frequently administered to a patient.
  • frequent administration via injection to maintain blood concentration of the physiologically active polypeptide causes severe pain to the patients and increases costs for treatment.
  • efforts have been made to maximize the efficacy of protein drugs by increasing blood stability of the protein drugs and maintaining the blood concentration thereof at a high level for a long period of time.
  • these long-acting formulations of protein drugs should not induce immune responses in patients while increasing the stability of the protein drugs and maintaining the titer of the drug at a sufficiently high level.
  • PEG polyethylene glycol
  • WO 2007/021129 A1 discloses a method for the mass production of a monomeric or dimeric immunoglobulin Fc region, free of initial methionine residues, using a recombinant expression vector comprising a nucleotide sequence coding for a recombinant immunoglobulin Fc region comprising an immunoglobulin Fc region linked at the N-terminus thereof to an immunoglobulin Fc region via a peptide bond.
  • WO 2017/052329 A1 discloses a complex composition, of which positional isomers are minimized by using a N-terminus of an immunoglobulin Fc region as a binding site when the immunoglobulin Fc region is used as a carrier. Also provided are a protein complex which is prepared by N-terminal-specific binding of immunoglobulin Fc region, thereby prolonging blood half-life of the physiologically active polypeptide, maintaining in vivo potency at a high level, and having no risk of immune responses, a preparation method thereof, and a pharmaceutical composition including the same for improving in vivo duration and stability of the physiologically active polypeptide.
  • the protein complex prepared by the present invention may be usefully applied to the development of long-acting formulations of various physiologically active polypeptide drugs.
  • An object of the present invention is to provide a novel intermediate for preparing a long-acting drug conjugate.
  • Another object of the present invention is to provide a composition for preparing a long-acting drug conjugate including the intermediate.
  • Another object of the present invention is to provide a method of preparing a long-acting drug conjugate using the intermediate.
  • Another object of the present invention is to provide a long-acting drug conjugate prepared by the preparation method.
  • One aspect of the present invention provides a novel intermediate.
  • the present invention provides a compound having a structure of Formula 1 below or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: [Formula 1] X-L1-O(CH 2 CH 2 O) n -L2-R wherein in Formula 1 above,
  • the present invention provides a compound having a structure of Formula 2 below or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: wherein in Formula 2, n is from 200 to 250.
  • the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE, or IgM.
  • the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.
  • the immunoglobulin Fc region is in a dimeric form.
  • the immunoglobulin Fc region comprises an amino acid sequence of SEQ ID NO: 10.
  • the compound has a size of 40 kDa to 250 kDa.
  • compositions in preparing a long-acting drug conjugate comprising a compound having a structure of Formula 1 below, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, wherein the drug is a physiologically active polypeptide: [Formula 1] X-L1-O(CH 2 CH 2 O) n -L2-R in Formula 1 above,
  • the physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, Gprotein-coupled receptors, interleukins, interleukin receptors, enzymes, interleukinbinding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, ⁇ -1 antitrypsin, albumin, ⁇ -lactalbumin, apolipoprotein-E, highly glycosylated erythropoietin,
  • R of the compound of the composition is linked to cysteine of the drug.
  • immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.
  • composition is used to prepare a long-acting drug conjugate without performing ultrafiltration/diafiltration in preparation of the long-acting drug conjugate.
  • Another aspect of the present invention provides a method for preparing a long-acting conjugate of a physiologically active polypeptide.
  • the preparation method comprises preparing a conjugate by linking a mono-PEGylated immunoglobulin Fc region, prepared by linking a linker of Formula 3 below to the N-terminus of an immunoglobulin Fc region comprising a hinge sequence that comprises an amino acid sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro), to a physiologically active polypeptide: [Formula 3] CHO-L1-O(CH 2 CH 2 O) n -L2-R wherein in Formula 3,
  • the mono-PEGylated immunoglobulin Fc region is prepared by linking the linker of Formula 3 above to the N-terminus of the immunoglobulin Fc region at a pH of 4.0 to 8.0 in the presence of a reducing agent.
  • the conjugate is prepared by linking the linker of the mono-PEGylated immunoglobulin Fc region to the physiologically active polypeptide at a pH of 5.5 to 8.0.
  • the preparing of the conjugate is performed by reacting the mono-PEGylated immunoglobulin Fc region with the physiologically active polypeptide in a molar ratio of 1:1 to 1:3.
  • the method comprises preparing a mono-PEGylated immunoglobulin Fc region by linking a linker of Formula 3 to the N-terminus of the immunoglobulin Fc region; and preparing a conjugate by linking the linker of the mono-PEGylated immunoglobulin Fc region prepared in the previous step to a physiologically active polypeptide.
  • the linker of the mono-PEGylated immunoglobulin Fc region is linked to a cysteine of the physiologically active polypeptide.
  • the method comprises preparing a mono-PEGylated immunoglobulin Fc region by linking a linker of Formula 3 to the N-terminus of an immunoglobulin Fc region; purifying the monoPEGylated immunoglobulin Fc region prepared in the previous step by anionexchange chromatography in a buffer solution with a pH of 6.0 to 8.5; and preparing a conjugate by linking the linker of the mono-PEGylated immunoglobulin Fc region purified in the previous step to a physiologically active polypeptide.
  • the method is performed without ultrafiltration/diafiltration after preparing the mono-PEGylated immunoglobulin Fc region.
  • the method further comprises purifying the conjugate by hydrophobic interaction chromatography.
  • L1 is a straight or branched-chain C 1 -C 6 alkylene
  • L2 is -a1-NHCO- or -a1-NHCO-a2-; in which a1 and a2 are each independently a straight or branched-chain C 1 -C 6 alkylene
  • n is from 200 to 250
  • R is maleimide
  • the linker has a structure of Formula 4 below: wherein in Formula 4, n is from 200 to 250.
  • the linker has a size of 1 kDa to 100 kDa.
  • the physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, Gprotein-coupled receptors, interleukins, interleukin receptors, enzymes, interleukinbinding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, ⁇ -1 antitrypsin, albumin, ⁇ -lactalbumin, apolipoprotein-E, highly glycosylated erythropoietin
  • the physiologically active polypeptide is a GLP-1/GIP/Glucagon trigonal agonist, glucagon, or an analog thereof.
  • the physiologically active polypeptide includes one of amino acid sequences of SEQ ID NOS: 1 to 6.
  • the hinge sequence comprises an amino acid sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro).
  • the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.
  • Another aspect of the present invention provides use of a composition in preparing a long-acting drug conjugate comprising a compound having a structure of Formula 2 below or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: wherein the drug is a physiologically active polypeptide: wherein in Formula 2 above, n is from 200 to 250.
  • Another aspect of the present invention provides a method for preparing a long-acting conjugate of a physiologically active polypeptide, the method comprising:
  • a long-acting drug conjugate may be prepared with a high yield although some of conventional purification processes are omitted, and thus productivity of the long-acting drug conjugate may be increased.
  • FIG. 1 shows results of analyzing a structure of a mono-PEGylated immunoglobulin Fc region by MALDI-TOF assay.
  • alanine Ala A arginine Arg, R asparagine Asn, N aspartic acid Asp, D cysteine Cys, C glutamic acid Glu, E glutamine Gln, Q glycine Gly, G histidine His, H isoleucine Ile, I leucine Leu, L lysine Lys, K methionine Met, M phenylalanine Phe, F proline Pro, P serine Ser, S threonine Thr, T tryptophan Trp, W tyrosine Tyr, Y valine Val, V
  • An aspect of the present invention provides a compound having a structure of Formula 1 below, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: [Formula 1] X-L1-O(CH 2 CH 2 O) n -L2-R In Formula 1 above,
  • the compound having a structure of Formula 1, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is a novel substance prepared for preparing a long-acting conjugate and may also be referred to as "intermediate” or “intermediate material” in the present application.
  • purification steps by ultrafiltration/diafiltration and hydrophobic interaction chromatography may be omitted, and effects on preparing the long-acting drug conjugate with a high yield may be obtained although the purification steps are omitted.
  • the intermediate is in a form in which an immunoglobulin Fc region is linked to a linker.
  • X is an immunoglobulin Fc region and comprises, at the N-terminus, a hinge sequence that comprises an amino acid sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro), and L1-O(CH 2 CH 2 O) n -L2-R may be a linker.
  • L1 is a straight or branched-chain C 1 -C 6 alkylene
  • L2 is -a1-NHCO- or -a1- NHCO-a2-; in which a1 and a2 are each independently a straight or branched-chain C 1 -C 6 alkylene
  • n is from 200 to 250
  • R is maleimide
  • the L1 is a site binding to the immunoglobulin Fc region and may be a straight or branched-chain C 1 -C 6 alkylene.
  • R is a site for linkage between the intermediate and a physiologically active polypeptide and, specifically, may include a reactive group (e.g., thiol, maleimide, aldehyde, and succinimidyl) capable of binding to a cysteine, or an amine group of the N-terminus, or a lysine residue of a physiologically active polypeptide.
  • a reactive group e.g., thiol, maleimide, aldehyde, and succinimidyl
  • the intermediate may have a structure of Formula 2 below:
  • n is from 200 to 250.
  • the intermediate has a size of 40 kDa to 250 kDa.
  • immunoglobulin Fc region refers to a region including a heavy chain constant domain 2 (CH2) and/or a heavy chain constant domain 3 (CH3) excluding the heavy chain and light chain variable domains of the immunoglobulin.
  • the immunoglobulin Fc region may be a component constituting a moiety of the long-acting drug conjugate of the present invention.
  • X is an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE, or IgM, more specifically, an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.
  • the immunoglobulin Fc region may include a particular hinge sequence at the N-terminus.
  • flankinge sequence refers to a site located at a heavy chain and forming a dimer of the immunoglobulin Fc region via an inter disulfide bond.
  • N-terminus refers to amino terminus of a protein or polypeptide and may include an amino acid residue located at the end of the amino terminus or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the end of the amino terminus.
  • the immunoglobulin Fc region of the present invention may include the hinge sequence at the N-terminus.
  • the hinge sequence of the present invention may consist of 3 to 12 amino acids including only one cysteine residue. More specifically, the hinge sequence of the present invention may have a sequence as follows: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Pro- Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro, Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys, Glu-Lys-Tyr-Gly-Pro-Pro-Cys, Glu-Lys-Tyr-Gly-Pro-Pro-C
  • X may be a dimer formed of two chain molecules of the immunoglobulin Fc region in the presence of the hinge sequence, and the intermediate of the present invention may be in a form in which one end of the linker is linked to one chain of the immunoglobulin Fc region in the dimeric form.
  • the immunoglobulin Fc region is in a dimeric form.
  • X of the present invention may be an immunoglobulin Fc region comprising an amino acid sequence of SEQ ID NO: 10.
  • the immunoglobulin Fc region of the present invention may be an extended Fc region including a part of or the entirety of a heavy chain constant domain 1 (CH1) and/or a light chain constant domain 1 (CL1) excluding the heavy chain and the light chain variable domains of the immunoglobulin, as long as the immunoglobulin Fc region has substantially identical or enhanced effects compared to the native type.
  • the immunoglobulin Fc region may be a region from which a considerably long part of the amino acid sequence corresponding to the CH2 and/or CH3 is removed.
  • the immunoglobulin Fc region of the present invention may include 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) a combination of one or more domains selected from CH1 domain, CH2 domain, CH3 domain, and CH4 domain and an immunoglobulin hinge region (or a part of the hinge region), or 6) a dimer of each domain of the heavy chain constant domain and the light chain constant domain.
  • the immunoglobulin Fc region of the present invention includes not only a naturally occurring amino acid sequence but also a sequence derivative thereof.
  • the amino acid sequence derivative refers to a sequence different from the naturally occurring amino acid sequence due to a deletion, insertion, non-conservative or conservative substitution, or any combination of one or more amino acids of the naturally occurring amino acid sequence.
  • amino acid residues known to be important in linkage at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 may be used as a suitable target for modification.
  • a site capable of forming a disulfide bond is deleted or certain amino acid residues are eliminated from the N-terminus of a native Fc form, and a methionine residue is added to the N-terminus of the native Fc form may be used.
  • a complement binding site such as a C1q binding site, may be deleted, and an antibody dependent cell mediated cytotoxicity (ADCC) site may be deleted.
  • ADCC antibody dependent cell mediated cytotoxicity
  • Amino acid exchanges in proteins and peptides which do not generally alter the activity of molecules, are known in the art ( H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979 ).
  • the most commonly occurring exchanges of amino acid residues are exchanges between Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/ Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • the Fc region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.
  • the above-described sequence derivatives of the Fc region are derivatives that have a biological activity equivalent to that of the immunoglobulin Fc region of the present invention or improved structural stability against heat, pH.
  • these immunoglobulin Fc regions may be obtained from native forms isolated from humans and other animals including cows, goats, swine, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof, obtained from transformed animal cells or microorganisms.
  • they may be obtained from a native immunoglobulin by isolating whole immunoglobulins from living humans or animals and treating them with a protease. Papain digests the native immunoglobulin into Fab and Fc regions and pepsin digests the native immunoglobulin into pF'c and F(ab) 2 fragments.
  • a human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.
  • the immunoglobulin Fc region of the present invention may have natural glycans or increased or decreased glycans compared to the natural type, or be in a deglycosylated form.
  • the increase, decrease, or removal of glycans of the immunoglobulin Fc may be achieved by any methods commonly used in the art such as a chemical method, an enzymatic method, and a genetic engineering method using a microorganism.
  • the immunoglobulin Fc region obtained by removing glycans shows a significant decrease in binding affinity to a complement c1q and a decrease in or loss of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and thus unnecessary immune responses are not induced thereby in living organisms.
  • a deglycosylated or aglycosylated immunoglobulin Fc region may be more suitable as a drug carrier in view of the objects of the present invention.
  • deglycosylation refers to a Fc region from which glycan is removed using an enzyme and the term “aglycosylation” refers to a Fc region that is not glycosylated and produced in prokaryotes, more specifically, E. coli.
  • the immunoglobulin Fc region may be derived from humans or animals such as cows, goats, swine, mice, rabbits, hamsters, rats, or guinea pigs. In a more specific embodiment, the immunoglobulin Fc region may be derived from humans.
  • the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, or IgM, or any combination or hybrid thereof.
  • the immunoglobulin Fc region is derived from IgG or IgM which are the most abundant proteins in human blood, and in an even more specific embodiment, it is derived from IgG known to enhance the half-lives of ligand-binding proteins.
  • the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4.
  • the term "combination" related to the immunoglobulin Fc region refers to formation of a linkage between a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin and a singlechain polypeptide of a different origin when a dimer or a multimer is formed. That is, a dimer or multimer may be prepared using two or more Fc fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.
  • hybrid means that sequences corresponding to two or more immunoglobulin Fc regions of different origins are present in a single-chain of an immunoglobulin constant domain.
  • a domain hybrid may be composed of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc and may further include a hinge region.
  • IgG may also be classified into IgG1, IgG2, IgG3 and IgG4 subclasses, which may be combined or hybridized in the present invention.
  • IgG2 and IgG4 subclasses Preferred are IgG2 and IgG4 subclasses, and most preferred is the Fc fragment of IgG4 rarely having effector functions such as complement dependent cytotoxicity (CDC).
  • linker refers to a moiety linking a drug (e.g., physiologically active polypeptide) to the immunoglobulin Fc region in the long-acting drug conjugate, and the linker may be a peptidyl linker or a non-peptidyl linker. Specifically, the linker may be represented by Formula 3 below: [Formula 3] CHO-L1-O(CH 2 CH 2 O) n -L2-R
  • the linker may include polyethylene glycol and has particular chemical structures at both ends of polyethylene glycol.
  • L1 may be a straight or branched-chain C 1 -C 6 alkylene
  • L2 may be -a1-NHCO- or -a1-NHCO-a2-; in which a1 and a2 may be each independently a straight or branched-chain C 1 -C 6 alkylene
  • n may be from 200 to 250
  • R may be maleimide
  • the linker may have a size of 1 kDa to 200 kDa, 1 kDa to 150 kDa, 1 kDa to 100 kDa, 1 kDa to 50 kDa, or 1 kDa to 10 kDa.
  • linker may have a structure of Formula 4 below:
  • n is from 200 to 250.
  • One end of the linker may be linked to the immunoglobulin Fc region, specifically, the N-terminus of the immunoglobulin Fc region, more specifically, the hinge sequence located at the N-terminus of the immunoglobulin Fc region, even more specifically, a proline residue of the hinge sequence, to form the intermediate.
  • the term "pharmaceutically acceptable” refers to a substance that may be effectively used for the intended use within the scope of pharmaco-medical decision without inducing excessive toxicity, irritation, or allergic responses.
  • the term "pharmaceutically acceptable salt” refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base.
  • a suitable acid may include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene2-sulfonic acid, and benzenesulfonic acid.
  • the salt derived from a suitable base may include alkali metals such as sodium and potassium, alkali earth metals such as magnesium, and ammonium.
  • the present invention comprises not only the compound or a pharmaceutically acceptable salt thereof, but also a solvate prepared therefrom.
  • the compound may be present in the form of an enantiomer (R or S isomer), racemate, or diastereomer, or any mixture thereof in the case of having an asymmetric carbon center (absent carbon) in a substituent thereof.
  • the compound may be present in the form of an exo or endo isomer in the case of having a bridged ring.
  • An aspect of the present invention provides a composition including a compound having a structure of Formula 1 below, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, wherein the drug is a physiologically active polypeptide: [Formula 1] X-L1-O(CH 2 CH 2 O) n -L2-R
  • L1 is a straight or branched-chain C 1 -C 6 alkylene
  • L2 is -a1-NHCO- or -a1-NHCO-a2-; in which a1 and a2 are each independently a straight or branched-chain C 1 -C 6 alkylene
  • n is from 200 to 250
  • R is maleimide
  • composition of the present invention comprises the intermediate and has a use for preparing a long-acting drug conjugate.
  • the composition of the present invention comprises the intermediate in which the linker is linked to the immunoglobulin Fc region
  • the composition of the present invention may be reacted with a physiologically active polypeptide such that the linker of the intermediate is linked to the physiologically active polypeptide, thereby preparing a long-acting drug conjugate.
  • the long-acting drug conjugate may be prepared via linkage between R of Formula 1 corresponding to one end of the linker and a cysteine, or an amine group such as the N-terminus, or a lysine residue of the physiologically active polypeptide.
  • any physiologically active polypeptide of the longacting drug conjugate that may be prepared using the composition may fall within the scope of the present invention regardless of type, size, origin, as long as the physiologically active polypeptide has pharmacological effects on disease.
  • the physiologically active polypeptide may include glucagon-like peptide1 (GLP-1), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, G-proteincoupled receptors, interleukins, interleukin receptors, enzymes, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastas
  • the physiologically active polypeptide may be glucagon-like peptide-1 (GLP-1), glucagon, insulin, enzyme, incretin, gastric inhibitory polypeptide (GIP), GLP-1/GIP dual agonist, or GLP-1/GIP/Glucagon triple agonist.
  • GLP-1 glucagon-like peptide-1
  • GIP gastric inhibitory polypeptide
  • GLP-1/GIP dual agonist GLP-1/GIP/Glucagon triple agonist
  • any physiologically active polypeptide including an amino acid residue or a reactive group capable of binding to the intermediate may be used regardless of types thereof to prepare the long-acting drug conjugate using the intermediate or the composition including the same of the present invention.
  • the immunoglobulin Fc region is derived [00179] from IgG1, IgG2, IgG3, or IgG4.
  • the long-acting drug conjugate may be prepared without performing ultrafiltration/diafiltration and one cycle of hydrophobic interaction chromatography may be optionally omitted.
  • purity of the long-acting drug conjugate may be 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.
  • the purity may be measured by any method well known in the art, specifically, by SE-HPLC, RP-HPLC, and IE-HPLC, but any method available in the art. More specifically, the purity of the long-acting drug conjugate prepared using the composition of the present invention may be 90% or more in SE-HPLC, 80% or more in RP-HPLC, and 85% or more in IE-HPLC.
  • composition of the present invention may further include a buffer, a stabilizer, a preservative, a salt, required to stabilize the intermediate and to prepare the long-acting drug conjugate.
  • kits for preparing a long-acting drug conjugate including the composition.
  • the kit may include a reagent, manual for preparing the long-acting drug conjugate.
  • Another aspect of the present invention provides a method for preparing a long-acting drug conjugate.
  • the preparation method of the present invention is a method for preparing a long-acting drug conjugate in which a drug is linked to an immunoglobulin Fc region via a linker, specifically, a method for preparing a long-acting drug conjugate by linking the intermediate to the drug.
  • the preparation method is characterized by sequentially performing i) linking a linker including polyethylene glycol (PEG) to an immunoglobulin Fc region, and ii) linking the linker, which is linked to the immunoglobulin Fc region, to a drug (e.g., a physiologically active polypeptide or protein). That is, the preparation method is characterized by performing steps in a particular order, i.e., performing a first step of preparing the intermediate by linking the linker including PEG to the immunoglobulin Fc region, and then performing a second step of linking the drug to the intermediate.
  • a linker including polyethylene glycol (PEG) to an immunoglobulin Fc region
  • a drug e.g., a physiologically active polypeptide or protein
  • the preparation method of the present invention may also be performed only by the second step of preparing the long-acting drug conjugate via a reaction between the intermediate or the composition for preparing the long-acting drug conjugate including the same and the drug, without performing the first step.
  • This preparation method may be referred to as "reverse order preparation method" in the present application.
  • the purification processes by ultrafiltration/diafiltration and hydrophobic interaction chromatography may be omitted and it was confirmed that the long-acting drug conjugate may be prepared with a high yield although the purification processes are omitted.
  • the linker is first linked to the physiologically active polypeptide and then linked to the immunoglobulin Fc region without forming the intermediate
  • the physiologically active polypeptide-linked linker e.g., polyethylene glycol
  • ultrafiltration/diafiltration is required as a separate process after the linker is linked to the physiologically active polypeptide and before the linked product is linked to the immunoglobulin Fc region to reduce the risk of aggregation that may occur due to low pH conditions (pH of about 3.0) of an equilibrium buffer and an elution buffer used for purification of the physiologically active polypeptide-linked linker and to adjust the pH conditions for reaction using an appropriate buffer.
  • a pH of a buffer used in purification of the immunoglobulin Fc region-linked linker is relatively high, and thus the ultrafiltration/diafiltration process may be omitted and then a process of linking the immunoglobulin Fc region-linked linker to the physiologically active polypeptide may be performed.
  • ultrafiltration/ diafiltration may not be performed after preparing a mono-PEGylated immunoglobulin Fc region.
  • a pH of a solution used to purify the mono-PEGylated immunoglobulin Fc region is not significantly different from a pH of a solution used for a subsequent reaction so that linkage to the drug may be performed without conducting the ultrafiltration/diafiltration.
  • the preparation method of the present invention may further include purifying the conjugate by hydrophobic interaction chromatography.
  • hydrophobic interaction chromatography may be performed only once or more than once in accordance with properties of the drug of the long-acting drug conjugate and type and size of the linker.
  • an amount of the expensive drug may be reduced but also an amount of unreacted immunoglobulin Fc regions may be reduced, so that the entire or a part of the purification process by hydrophobic interaction chromatography may be omitted to obtain effects on reducing raw materials required for preparation of the long-acting drug conjugate and costs therefor compared to the conventional method.
  • the ultrafiltration/diafiltration and hydrophobic interaction chromatography processes which have been performed in the conventional method for preparing the long-acting drug conjugate, are omitted and only the final purification process (e.g., one cycle of hydrophobic interaction chromatography) is performed in the preparation method of the present invention, it is advantageous in that a purity of the final conjugate obtained by the present invention is maintained compared to that of the conventional preparation method. That is, according to the preparation method of the present invention, the final purity may be maintained although some of the purification processes are omitted so that productivity of the long-acting drug conjugate may be improved.
  • the purity of the long-acting drug conjugate according to the present invention may be measured by any method well known in the art and examples of the method may be SE-HPLC, RP-HPLC, and IE-HPLC.
  • the final purity of the long-acting drug conjugate may be 90% or more, specifically, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.
  • the monoPEGylated immunoglobulin Fc region is prepared first and then linked to the physiologically active polypeptide, so that the long-acting drug conjugate may be prepared with a higher yield compared to the conventional method in terms of not only the physiologically active polypeptide but also the immunoglobulin Fc region.
  • the yield of the long-acting drug conjugate obtained by the preparation method of the present invention was increased twice or more compared to the yield of the long-acting drug conjugate obtained by the conventional method.
  • the preparation method of the present invention relates to a method for preparing a long-acting drug conjugate including preparing a conjugate by linking a mono-PEGylated immunoglobulin Fc region, which is prepared by linking a linker of Formula 3 below to the N-terminus of an immunoglobulin Fc region comprising a hinge sequence that comprises an amino acid sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro), to a physiologically active polypeptide: [Formula 3] CHO-L1-O(CH 2 CH 2 O) n -L2-R
  • the step of preparing the conjugate according to the preparation method of the present invention may be performed by reacting the physiologically active polypeptide in an amount equivalent to or more than an amount of the monoPEGylated immunoglobulin Fc region, and specifically, a molar ratio of monoPEGylated immunoglobulin Fc region : physiologically active polypeptide may be from 1:1 to 1:3.
  • the term "mono-PEGylated immunoglobulin Fc region” refers to an intermediate material that is produced in the middle of the method for preparing the long-acting drug conjugate according to the present invention in which one linker including one polyethylene glycol is linked to the immunoglobulin Fc region. That is, in the present invention, the "mono-PEGylated immunoglobulin Fc region" may be used interchangeably with “intermediate” or “intermediate material”.
  • the immunoglobulin Fc region may be an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.
  • the immunoglobulin Fc region is an immunoglobulin Fc region comprising a hinge sequence that comprises an amino acid sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro).
  • the "long-acting drug conjugate" of the present invention refers to a drug conjugate having a structure in which a drug (physiologically active polypeptide) having a pharmacological activity in the body is linked to an immunoglobulin Fc region via a linker and an increased half-life.
  • the long-acting drug conjugate may be one in which the intermediate or monoPEGylated immunoglobulin Fc region is linked to the drug.
  • the drug is not limited to particular substances as long as the drug has preventive, therapeutic, or alleviating effects on a certain disease and may be a natural or non-natural protein, enzyme, antibody, compound. More specifically, the drug may be a physiologically active polypeptide or protein, even more specifically, the physiologically active polypeptide may be glucagon-like peptide-1 (GLP-1), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, G-proteincoupled receptors, interleukins, interleukin receptors, enzymes, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor
  • the physiologically active polypeptide may be GLP1/GIP/Glucagon trigonal agonist, glucagon, or a analog thereof. Even more specifically, the physiologically active polypeptide may include, essentially consist of, or consist of one of amino acid sequences of SEQ ID NOS: 1 to 6.
  • variant refers to a peptide having an amino acid sequence in which one or more amino acids are different from those of a native physiologically active polypeptide while retaining the same functions as those of the native physiologically active polypeptide, and the variant may be prepared by substitution, addition, deletion, modification, or any combination of some amino acids of the amino acid sequence of the native physiologically active polypeptide.
  • derivative refers to a peptide, a peptide analog, or a peptidomimetic obtained by modifying one or more amino acids of the native physiologically active polypeptide by addition, deletion, or substitution to have similar activity to that of the native physiologically active polypeptide.
  • fragment refers to a form obtained by adding/ deleting one or more amino acids to/from the N-terminus or the C-terminus, and the added amino acid may be any amino acid that does not exist in nature (e.g., D-amino acid).
  • the methods for preparing the variant, derivative, and fragment of the physiologically active polypeptide may be used independently or in combination.
  • any physiologically active polypeptide having one or more different amino acids in the amino acid sequence and deamination of an amino acid residue at the N-terminus may be included therein.
  • the derivative of the physiologically active polypeptide includes biosimilar and biobetter forms.
  • the biosimilar may be any biosimilar enzyme available in the long-acting drug conjugate of the present invention although there is a difference between a known enzyme and a host for its expression, a difference in glycosylation feature and the degree thereof, and a difference in the degree of substitution in a particular amino acid residue of the corresponding enzyme in light of the standard sequence where the degree of substitution is not 100% substitution.
  • the physiologically active polypeptide and the variant, derivative and fragment thereof may be produced from animal cells, E. coli, yeast, insect cells, plant cells, living animals, via genetic recombination, and any commercially available physiologically active polypeptides, and variants, derivatives, and fragments thereof may also be used.
  • physiologically active polypeptide, and the variant, derivative and fragment thereof may include an amino acid sequence having a homology of at least 80%, specifically, at least 90%, more specifically, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and the physiologically active polypeptide, and the variant, derivative, and fragment thereof may be obtained from microorganisms by genetic recombination technologies or commercially available.
  • the term "homology" refers to the degree of similarity between amino acid sequences of a wild-type protein or nucleotide sequences encoding the same and includes a sequence identical to the amino acid sequence or nucleotide sequence of the present invention by the above-described percentage or more.
  • the homology may be determined by comparing the sequences via visual observation but may also be determined using a bioinformatic algorithm, which provides analysis results of a degree of homology by aligning sequences to be compared. The homology between the two amino acid sequences may be indicated in percentage.
  • Useful automated algorithms may be used in GAP, BESTFIT, FASTA, and TFASTA computer software modules of Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI, USA).
  • the automated alignment algorithms in the modules include the Needleman & Wunsch algorithm, the Pearson & Lipman algorithm, and the Smith & Waterman sequence algorithm.
  • Other useful algorithms and homology determinations on alignment are automated in software such as FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.
  • sequences of the physiologically active polypeptide, and the variant, derivative, and fragment thereof, and nucleotide sequences encoding the same may be obtained from known database of the NCBI GenBank.
  • the amino acids substituted or added may be not only 20 amino acids commonly found in human proteins but also atypical or non-naturally occurring amino acids.
  • Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPep Inc. and Genzyme pharmaceuticals.
  • the peptides including theses amino acids and typical peptide sequences may be synthesized and purchased from commercial suppliers, e.g., American Peptide Company, Bachem (USA) or Anygen (Korea).
  • physiologically active polypeptide, and the variant, derivative, and fragment thereof according to the present invention may be in a varied form where the N-terminus and/or C-terminus is chemically modified or protected by organic groups, or amino acids may be added to the termini of the peptide, for protection from proteases in vivo while increasing stability thereof.
  • the N-terminus may be acetylated and/or the C-terminus may be amidated to remove the charges.
  • the peptide according to the present invention includes all of those in the form of the peptide itself, a salt thereof (e.g., a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Also, the peptide may be in any pharmaceutically acceptable form.
  • the type of the salt is not particularly limited. However, the salt is preferably in a form safe and effective to an individual, e.g., a mammal.
  • solvate refers to a complex of the peptide or a salt thereof according to the present invention and a solvent molecule.
  • the method for preparing the long-acting drug conjugate according to the present invention may be a method for preparing a conjugate in which a physiologically active polypeptide is linked to an immunoglobulin Fc region via a linker.
  • linkage between the linker and the immunoglobulin Fc region may be formed by a covalent bond or a non-covalent bond between one end of the linker and the N-terminus of the immunoglobulin Fc region, but binding sites or methods for the linkage are not particularly limited.
  • the mono-PEGylated immunoglobulin Fc region may be prepared by linking a proline at the N-terminus of the immunoglobulin Fc region to a -CHO group of the linker.
  • the linker may have a structure of Formula 4 below:
  • n is from 200 to 250.
  • the linker has a size of 1 kDa to 100 kDa.
  • the mono-PEGylated immunoglobulin Fc region may have a structure of Formula 2 below.
  • the preparation method of the present invention may be performed by linking the physiologically active polypeptide to one end of the mono-PEGylated immunoglobulin Fc region having the structure of Formula 2.
  • Fc region may be linked to the physiologically active polypeptide, specifically, a -SH group or an amino acid containing a -SH group, or a cysteine of the physiologically active polypeptide.
  • the long-acting drug conjugate is prepared according to the preparation method of the present invention in which the mono-PEGylated immunoglobulin Fc region is prepared first and linked to the physiologically active polypeptide, it was confirmed that the purity of the final conjugate may be maintained with an increased yield compared to the conventional preparation method although the ultrafiltration/ diafiltration and hydrophobic interaction chromatography processes are omitted and only the final purification process (e.g., one cycle of hydrophobic interaction chromatography) is performed in the preparation method according to the present invention.
  • Another aspect of the present invention provides a long-acting drug conjugate prepared by the above-described method.
  • the long-acting drug conjugate prepared by the preparation of the present invention has an increased half-life compared to the physiologically active polypeptide that is not linked to the linker or the immunoglobulin Fc region, advantageous effects on preparation of drugs may be obtained.
  • the long-acting drug conjugate prepared by the preparation method of the present invention may be used in preparation of drugs or compositions for the purposes of prevention, treatment, and alleviation of diseases.
  • a PEGylated physiologically active polypeptide was linked to an immunoglobulin Fc region to prepare a long-acting conjugate.
  • GLP1/GIP/Glucagon trigonal agonist analog 1 SEQ ID NO: 1
  • a linker containing PEG maleimide-10 kDa-PEG-aldehyde
  • the reaction was performed in a 50 mM Tris buffer containing isopropanol (pH of 7.5, 6°C ⁇ 4°C).
  • the reaction solution was diluted with an equilibrium buffer including sodium citrate and ethanol to a total volume of 20 times and purified.
  • the mono-PEGylated GLP-1/GIP/Glucagon trigonal agonist analog 1 was purified using an SP High Performance column (GE Healthcare, cation-exchange chromatography) using a solution including sodium citrate and ethanol and a potassium chloride concentration gradient.
  • the buffer solution was replaced with a 0.1 M potassium phosphate solution through ultrafiltration/diafiltration (UF/DF), followed by concentration to recover a resultant with a final concentration of about 3 g/L or more.
  • UF/DF ultrafiltration/diafiltration
  • the mono-PEGylated GLP-1/GIP/Glucagon trigonal agonist analog 1 prepared as described above was linked to an immunoglobulin Fc region to prepare a long-acting conjugate as follows.
  • the mono-PEGylated GLP-1/GIP/Glucagon trigonal agonist analog 1 was reacted with the immunoglobulin Fc region in a molar ratio of 1:2 at a temperature of 6°C ⁇ 4°C for about 12 hours with a total protein concentration (GLP-1/GIP/Glucagon trigonal agonist analog 1 and immunoglobulin Fc region) of 30 g/L.
  • reaction solution was purified using a Butyl 4 Fast Flow column (GE Healthcare, hydrophobic interaction chromatography).
  • a Tris buffer and sodium chloride were added to the reaction solution, and the reaction solution was purified using a solution including a Bis-Tris and a sodium chloride concentration gradient.
  • Glucagon analog 1 was reacted with a linker containing PEG (maleimide-10 kDa-PEG-aldehyde) (Formula 1) for about 1 hour in a molar ratio of 1:1.3 with a Glucagon analog 1 concentration of 3 g/L. Specifically, the reaction was performed in a 50 mM Tris buffer containing isopropanol (pH of 7.3).
  • the reaction solution was diluted with an equilibrium buffer including sodium citrate and ethanol to a total volume of 20 times and purified.
  • the mono-PEGylated Glucagon analog 1 was purified using an SP High Performance column (GE Healthcare, cation exchange chromatography) using a solution including sodium citrate and ethanol and a potassium chloride concentration gradient.
  • the buffer solution was replaced with a 0.1 M potassium phosphate solution through ultrafiltration/diafiltration (UF/DF), followed by concentration to recover a resultant with a final concentration of 3 g/L or more.
  • the mono-PEGylated Glucagon analog 1 prepared as described above was linked to an immunoglobulin Fc region to prepare a long-acting conjugate as follows.
  • the mono-PEGylated Glucagon analog 1 was reacted with the immunoglobulin Fc region in a molar ratio of 1:5 at a temperature of 6°C ⁇ 4°C for about 12 hours with a total protein concentration (Glucagon analog 1 and immunoglobulin Fc region) of 20 g/L.
  • reaction solution was purified using a Butyl 4 Fast Flow column (GE Healthcare, hydrophobic interaction chromatography).
  • a Tris buffer and sodium chloride were added to the reaction solution, and the reaction solution was purified using a solution including Bis-Tris and a sodium chloride concentration gradient.
  • the present inventors have developed a process capable of efficiently producing the conjugate with a high purity by omitting the membrane filtration process and the purification process (hydrophobic interaction chromatography, Butyl 4 Fast Flow) from the process of preparing the conjugate according to the above-described Comparative Examples 1 and 2 as follows.
  • an immunoglobulin Fc region (49.8 kDa) having a hinge region with a Pro-Ser-Cys-Pro sequence at the N-terminus
  • the immunoglobulin Fc region was reacted with a linker containing PEG (structure of Formula 4, 10 kDa) in a molar ratio (immunoglobulin Fc region : PEG-containing linker) of 1:1 with an immunoglobulin Fc region concentration of 50 g/L at 6°C ⁇ 4°C for about 4 hours.
  • the reaction was performed in a composition including a 5 mM Bis-Tris buffer (pH 6.5) and potassium phosphate, and 10 mM NaCNBH 3 (sodium cyanoborohydride) was added thereto as a reducing agent.
  • the reaction solution was diluted with the Bis-Tris buffer and purified.
  • the monoPEGylated immunoglobulin Fc region was purified using a CaptoQ ImpRes column (GE Healthcare, anion-exchange chromatography) using a Bis-Tris buffer and a sodium chloride concentration gradient.
  • the mono-PEGylated immunoglobulin Fc region prepared in Example 1-1 was structurally analyzed by MALDI-TOF and Peptide mapping.
  • MALDITOF the resultant was identical to an expected molecular weight of the monoPEGylated immunoglobulin Fc region ( FIG. 1 ), and as a result of Peptide mapping, it was confirmed that over 90% of PEG was PEGylated at the N-terminus of the immunoglobulin Fc region.
  • Long-acting conjugates were prepared as follows by linking the monoPEGylated immunoglobulin Fc region prepared in Example 1-1 to various physiologically active peptides.
  • a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-1/GIP/Glucagon trigonal agonist analog 1) was prepared via peptide conjugation of the GLP-1/GIP/Glucagon trigonal agonist analog 1 (SEQ ID NO: 1), after anionexchange chromatography of Example 1-1, without performing ultrafiltration/ diafiltration.
  • the mono-PEGylated immunoglobulin Fc region was reacted with the GLP-1/GIP/Glucagon trigonal agonist analog 1 in a molar ratio of 1:1 with a GLP-1/GIP/Glucagon trigonal agonist analog 1 concentration of 0.2 g/L at 6°C ⁇ 4°C for about 2 hours.
  • the reaction was performed in a Tris-Cl buffer (6°C ⁇ 4°C) including isopropanol.
  • the purity was confirmed to be 90% or more in SE-HPLC, 80% or more in RP-HPLC, and 70% or more in IE-HPLC.
  • the eluted immunoglobulin Fc region-PEG-containing linker-GLP1/GIP/Glucagon trigonal agonist analog 1 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was 90% or more in SE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.
  • a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-1/GIP/Glucagon trigonal agonist analog 2) was prepared via peptide conjugation of the GLP-1/GIP/Glucagon trigonal agonist analog 2 (SEQ ID NO: 2), after anionexchange chromatography of Example 1-1, without performing ultrafiltration/ diafiltration.
  • the mono-PEGylated immunoglobulin Fc region was reacted with the GLP-1/GIP/Glucagon trigonal agonist analog 2 in a molar ratio of 1:1 with a GLP-1/GIP/Glucagon trigonal agonist analog 2 concentration of 0.2 g/L at 6°C ⁇ 4°C for about 2 hours.
  • the reaction was performed in a Tris-Cl buffer (6°C ⁇ 4°C) including isopropanol.
  • the purity of the long-acting conjugate including the GLP-1/GIP/Glucagon trigonal agonist analog 2 was confirmed to be 90% or more in SE-HPLC, 80% or more in RP-HPLC, and 70% or more in IE-HPLC.
  • the eluted immunoglobulin Fc region-PEG-containing linker-GLP1/GIP/Glucagon trigonal agonist analog 2 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was 90% or more in SE-HPLC, 90% or more in RP-HPLC, and 80% or more in IE-HPLC.
  • a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-1/GIP/Glucagon trigonal agonist analog 3) was prepared via peptide conjugation of the GLP-1/GIP/Glucagon trigonal agonist analog 3 (SEQ ID NO: 3), after anion exchange chromatography of Example 1-1, without performing ultrafiltration/ diafiltration.
  • the mono-PEGylated immunoglobulin Fc region was reacted with the GLP-1/GIP/Glucagon trigonal agonist analog 3 in a molar ratio of 1:1 with a GLP-1/GIP/Glucagon trigonal agonist analog 3 concentration of 0.2 g/L at 6°C ⁇ 4°C for about 2 hours.
  • the reaction was performed in a Tris-Cl buffer (6°C ⁇ 4°C) including isopropanol.
  • the purity was confirmed to be 90% or more in SE-HPLC, 80% or more in RP-HPLC, and 70% or more in IEHPLC.
  • the eluted immunoglobulin Fc region-PEG-containing linker-GLP1/GIP/Glucagon trigonal agonist analog 3 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was 90% or more in SE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.
  • a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-Glucagon analog 1) was prepared via peptide conjugation of Glucagon analog 1 (SEQ ID NO: 4), after anion-exchange chromatography of Example 1-1, without performing ultrafiltration/diafiltration.
  • the mono-PEGylated immunoglobulin Fc region was reacted with Glucagon analog 1 in a molar ratio of 1:1 with a Glucagon analog 1 concentration of 0.2 g/L at 6°C ⁇ 4°C for about 2 hours.
  • the reaction was performed in a Tris-Cl buffer (6°C ⁇ 4°C) including isopropanol.
  • the purity of the immunoglobulin Fc region-PEG containing linker-Glucagon analog 1 was confirmed to be 90% or more in SE-HPLC, 70% or more in RP-HPLC, and 70% or more in IE-HPLC.
  • the eluted immunoglobulin Fc region-PEG-containing linker-Glucagon analog 1 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was 90% or more in SE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.
  • a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-Glucagon analog 2) was prepared via peptide conjugation of Glucagon analog 2 (SEQ ID NO: 5), after anion-exchange chromatography of Example 1-1, without performing ultrafiltration/diafiltration.
  • the mono-PEGylated immunoglobulin Fc region was reacted with Glucagon analog 2 in a molar ratio of 1:1 with a Glucagon analog 2 concentration of 0.2 g/L at 6°C ⁇ 4°C for about 2 hours.
  • the reaction was performed in a Tris-Cl buffer (6°C ⁇ 4°C) including isopropanol.
  • the purity of the immunoglobulin Fc region-PEG containing linker-Glucagon analog 2 was confirmed to be 90% or more in SE-HPLC, 70% or more in RP-HPLC, and 70% or more in IE-HPLC.
  • the eluted immunoglobulin Fc region-PEG-containing linker-Glucagon analog 2 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was 90% or more in SE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.
  • a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-Glucagon analog 3) was prepared via peptide conjugation of Glucagon analog 3 (SEQ ID NO: 6), after anion-exchange chromatography of Example 1-1, without performing ultrafiltration/diafiltration.
  • the mono-PEGylated immunoglobulin Fc region was reacted with Glucagon analog 3 in a molar ratio of 1:1 with a Glucagon analog 3 concentration of 0.2 g/L at 6°C ⁇ 4°C for about 2 hours.
  • the reaction was performed in a Tris-Cl buffer (6°C ⁇ 4°C) including isopropanol.
  • the purity of the immunoglobulin Fc region-PEG-containing linker-Glucagon analog 3 was confirmed to be 90% or more in SE-HPLC, 70% or more in RP-HPLC, and 70% or more in IE-HPLC.
  • the eluted immunoglobulin Fc region-PEG-containing linker-Glucagon analog 3 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was 90% or more in SE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.
  • Tables 1 and 2 show comparison results between the methods the comparative examples and examples.

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Claims (26)

  1. Eine Verbindung mit einer Struktur der nachstehenden Formel 1 oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon:

            [Formel 1]     X-L1-O(CH2CH2O)n-L2-R

    wobei in der oberen Formel 1,
    X eine Immunglobulin-Fc-Region ist und am N-Terminus eine hinge Sequenz umfasst, die eine Aminosäuresequenz der SEQ ID NO: 9 (Pro-Ser-Cys-Pro) umfasst;
    L1 ist ein geradkettiges oder verzweigtes C1-C6 Alkylen und ist mit dem N-terminalen Prolinrest (Pro) von SEQ ID NO: 9 verbunden;
    L2 ist -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2- oder - b1-COO-b2-, wobei a1, a2, b1 und b2 jeweils unabhängig voneinander ein geradkettiges oder verzweigtes C1-C6 Alkylen sind;
    n ist von 10 bis 2.400; und
    R aus der Gruppe ausgewählt ist, bestehend aus 2,5-Dioxopyrrolidinyl, 2,5-Dioxopyrrolyl, Aldehyd, Maleimid, C6-C20 Aryldisulfid, C5-C20 Heteroaryldisulfid, Vinylsulfon, Thiol, halogeniertes Acetamid, Succinimid, p-Nitrophenylcarbonat und Thioester.
  2. Die Verbindung oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon nach Anspruch 1, wobei in Formel 1,
    L1 ein geradkettiges oder verzweigtes C1-C6 Alkylen ist;
    L2 -a1-NHCO- oder -a1-NHCO-a2- ist, wobei a1 und a2 jeweils unabhängig voneinander ein geradkettiges oder verzweigtes C1-C6 Alkylen sind;
    n ist von 200 bis 250; und
    R ist Maleimid.
  3. Eine Verbindung mit einer Struktur der nachstehenden Formel 2 oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon:
    Figure imgb0023
     wobei in der oberen Formel 2, n von 200 bis 250 ist.
  4. Die Verbindung oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon nach Anspruch 1, wobei die Immunglobulin-Fc-Region von IgG, IgA, IgD, IgE oder IgM abgeleitet ist.
  5. Die Verbindung oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon nach Anspruch 4, wobei die Immunglobulin-Fc-Region von IgG1, IgG2, IgG3 oder IgG4 abgeleitet ist.
  6. Die Verbindung oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon nach Anspruch 1, wobei die Immunglobulin-Fc-Region eine dimere Form hat.
  7. Die Verbindung oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon nach Anspruch 1, wobei die Immunglobulin-Fc-Region eine Aminosäuresequenz der SEQ ID NO: 10 umfasst.
  8. Die Verbindung oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon nach Anspruch 1, wobei die Verbindung eine Größe von 40 kDa bis 250 kDa hat.
  9. Verwendung einer Zusammensetzung zur Herstellung eines lang wirkenden Wirkstoffkonjugats, wobei die Zusammensetzung eine Verbindung mit einer Struktur der nachstehenden Formel 1 oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon umfasst,
    wobei der Wirkstoff ein physiologisch aktives Polypeptid ist:

            [Formel 1]     X-L1-O(CH2CH2O)n-L2-R

    in der oberen Formel 1,
    ist X eine Immunglobulin-Fc-Region und umfasst am N-Terminus eine hinge Sequenz, die eine Aminosäuresequenz der SEQ ID NO: 9 (Pro-Ser-Cys-Pro) umfasst;
    L1 ist ein geradkettiges oder verzweigtes C1-C6 Alkylen und ist mit dem N-terminalen Prolinrest (Pro) von SEQ ID NO: 9 verbunden;
    L2 ist -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2- oder - b1-COO-b2-, wobei a1, a2, b1 und b2 jeweils unabhängig voneinander ein geradkettiges oder verzweigtes C1-C6 Alkylen sind;
    n ist von 10 bis 2.400; und
    R ist aus der Gruppe ausgewählt, bestehend aus 2,5-Dioxopyrrolidinyl, 2,5-Dioxopyrrolyl, Aldehyd, Maleimid, C6-C20 Aryldisulfid, C5-C20 Heteroaryldisulfid, Vinylsulfon, Thiol, halogeniertes Acetamid, Succinimid, p-Nitrophenylcarbonat und Thioester.
  10. Verwendung nach Anspruch 9, wobei in der oberen Formel 1,
    L1 ein geradkettiges oder verzweigtes C1-C6 Alkylen ist;
    L2 -a1-NHCO- oder -a1-NHCO-a2- ist, wobei a1 und a2 jeweils unabhängig voneinander ein geradkettiges oder verzweigtes C1-C6 Alkylen sind;
    n ist von 200 bis 250; und
    R ist Maleimid.
  11. Verwendung nach Anspruch 9, wobei das physiologisch aktive Polypeptid ausgewählt ist aus der Gruppe bestehend aus Glukagon-ähnlichem Peptid-1 (GLP-1), Granulozyten-Kolonie-stimulierendem Faktor (G-CSF), menschlichem Wachstumshormon (hGH), Erythropoietin (EPO), Glukagon, Insulin, Wachstumshormon-Releasing-Hormon, Wachstumshormon-Releasing-Peptid, Interferone, Interferon-Rezeptoren, G-Protein-gekoppelte Rezeptoren, Interleukine, Interleukin-Rezeptoren, Enzyme, Interleukin-bindendes Protein, Zytokin-bindendes Protein, Makrophagen-Aktivierungsfaktor, Makrophagen-Peptid, B-Zell-Faktor, T-Zell-Faktor, Protein A, Allergie-Inhibitor, Zellnekrose-Glykoprotein, Immunotoxin, Lymphotoxin, Tumor-Nekrose-Faktor, Tumorsuppressor, Metastasen-Wachstumsfaktor, α-1-Antitrypsin, Albumin, α-Lactalbumin, Apolipoprotein-E, hochglykosyliertes Erythropoietin, Angiopoietine, Hämoglobin, Thrombin, Thrombinrezeptor-aktivierendes Peptid, Thrombomodulin, Blutfaktoren VII, VIIa, VIII, IX und XIII, Plasminogen-aktivierender Faktor, Fibrin-bindendes Peptid, Urokinase, Streptokinase, Hirudin, Protein C, C-reaktives Protein, Lenin-Inhibitor, Kollagenase-Inhibitor, Superoxiddismutase, Leptin, Thrombozyten-Wachstumsfaktor, Epithel-Wachstumsfaktor, epidermaler Wachstumsfaktor, Angiostatin, Angiotensin, Knochenwachstumsfaktor, Knochen stimulierendes Protein, Calcitonin, Atriopeptin, Knorpel induzierender Faktor, Elcatonin, Bindegewebe aktivierender Faktor, Inhibitor des Gewebefaktor-Signalwegs, Follikel stimulierendes Hormon, luteinisierendes Hormon, luteinisierendes Hormon freisetzendes Hormon, Nervenwachstumsfaktor, Nebenschilddrüsenhormon, Relaxin, Sekretin, Somatomedin, insulinähnlicher Wachstumsfaktor, Nebennierenrindenhormon, Cholecystokinin, Pankreas-Polypeptid, Gastrin-Releasing-Peptid, Corticotropin-Releasing-Faktor, schilddrüsenstimulierendes Hormon, Autotaxin, Lactoferrin, Myostatin, Inkretine, gastrisches inhibitorisches Polypeptid (GIP), GLP-1/GIP-Dualagonist, trigonaler GLP-1/GIP/Glukagon-Agonist, Zelloberflächenantigene, von Viren abgeleitete Impfstoffantigene, monoklonale Antikörper, polyklonale Antikörper und Antikörperfragmente.
  12. Verwendung nach Anspruch 9, wobei R in der Verbindung der Zusammensetzung mit Cystein des Wirkstoffs verbunden ist.
  13. Verwendung nach Anspruch 9, wobei die Fc-Region des Immunglobulins von IgG1, IgG2, IgG3 oder IgG4 abgeleitet ist.
  14. Verwendung nach Anspruch 9, wobei die Zusammensetzung zur Herstellung eines lang wirkenden Wirkstoffkonjugats verwendet wird, ohne dass eine Ultrafiltration/Diafiltration bei der Herstellung des lang wirkenden Wirkstoffkonjugats durchgeführt wird.
  15. Ein Verfahren zur Herstellung eines lang wirkenden Konjugats eines physiologisch aktiven Polypeptids, wobei das Verfahren umfasst:
    Herstellung eines Konjugats durch Linking einer mono-PEGylierten Immunglobulin-Fc-Region, hergestellt durch Linking eines Linkers der nachstehenden Formel 3 an den N-Terminus einer Immunglobulin-Fc-Region, die eine hinge Sequenz umfasst, die eine Aminosäuresequenz der SEQ ID NO: 9 (Pro-Ser-Cys-Pro) umfasst, an ein physiologisch aktives Polypeptid:

            [Formel 3]     CHO-L1-O(CH2CH2O)n-L2-R

    wobei in der obigen Formel 3,
    L1 ein geradkettiges oder verzweigtes C1-C6 Alkylen ist und mit dem N-terminalen Prolinrest (Pro) von SEQ ID NO: 9 verbunden ist;
    L2 -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2- oder -b1-COO-b2- ist, wobei a1, a2, b1 und b2 jeweils unabhängig voneinander ein geradkettiges oder verzweigtes C1-C6 Alkylen sind;
    n ist von 10 bis 2400; und;
    R ist ausgewählt aus der Gruppe bestehend aus 2,5-Dioxopyrrolidinyl, 2,5-Dioxopyrrolyl, Aldehyd, Maleimid, C6-C20 Aryldisulfid, C5-C20 Heteroaryldisulfid, Vinylsulfon, Thiol, halogeniertes Acetamid, Succinimid, p-Nitrophenylcarbonat und Thioester.
  16. Das Verfahren nach Anspruch 15, wobei die mono-PEGylierte Immunglobulin-Fc-Region durch Linking des Linkers der obigen Formel 3 an den N-Terminus der Immunglobulin-Fc-Region bei einem pH von 4,0 bis 8,0 in Gegenwart eines Reduktionsmittels hergestellt wird;
    oder
    wobei das Konjugat durch Linking des Linkers der mono-PEGylierten Immunglobulin-Fc-Region an das physiologisch aktive Polypeptid bei einem pH-Wert von 5,5 bis 8,0 hergestellt wird;
    oder
    wobei die Herstellung des Konjugats durch Reagieren der mono-PEGylierten Immunglobulin-Fc-Region mit dem physiologisch aktiven Polypeptid in einem molaren Verhältnis von 1:1 bis 1:3 durchgeführt wird.
  17. Das Verfahren nach Anspruch 15, wobei das Verfahren umfasst:
    Herstellung einer mono-PEGylierten Immunglobulin-Fc-Region durch Linking eines Linkers der Formel 3 an den N-Terminus der Immunglobulin-Fc-Region; und
    Herstellung eines Konjugats durch Linking des Linkers der im vorherigen Schritt hergestellten mono-PEGylierten Immunglobulin-Fc-Region an ein physiologisch aktives Polypeptid.
  18. Das Verfahren nach Anspruch 17, wobei der Linker der mono-PEGylierten Immunglobulin-Fc-Region an einen Cysteinrest des physiologisch aktiven Polypeptids gebunden ist;
    oder
    wobei das Verfahren umfasst:
    Herstellung einer mono-PEGylierten Immunglobulin-Fc-Region durch Linking eines Linkers der Formel 3 an den N-Terminus einer Immunglobulin-Fc-Region; Reinigen der im vorhergehenden Schritt hergestellten mono-PEGylierten Immunglobulin-Fc-Region durch Anionenaustauschchromatographie in einer Pufferlösung mit einem pH-Wert von 6,0 bis 8,5; und
    Herstellung eines Konjugats durch Linking des Linkers der im vorherigen Schritt gereinigten mono-PEGylierten Immunglobulin-Fc-Region an ein physiologisch aktives Polypeptid;
    oder
    wobei das Verfahren ohne Ultrafiltration/Diafiltration nach Herstellung der mono-PEGylierten Immunglobulin-Fc-Region durchgeführt wird.
  19. Das Verfahren nach Anspruch 15, ferner umfassend die Reinigung des Konjugats durch hydrophobe Wechselwirkungschromatographie.
  20. Das Verfahren nach Anspruch 15, wobei in Formel 3,
    L1 ein geradkettiges oder verzweigtes C1-C6 Alkylen ist; L2 -a1-NHCO- oder -a1-NHCO-a2- ist, wobei a1 und a2 jeweils unabhängig voneinander ein geradkettiges oder verzweigtes C1-C6 Alkylen sind;
    n ist von 200 bis 250; und
    R ist Maleimid.
  21. Das Verfahren nach Anspruch 15, wobei der Linker eine Struktur der nachstehenden Formel 4 hat:
    Figure imgb0024
     wobei in der obigen Formel 4 n von 200 bis 250 ist; und/oder wobei der Linker eine Größe von 1 kDa bis 100 kDa hat.
  22. Das Verfahren nach Anspruch 15, wobei das physiologisch aktive Polypeptid ausgewählt ist aus der Gruppe bestehend aus Glukagon-ähnlichem Peptid-1 (GLP-1), Granulozyten-Kolonie-stimulierendem Faktor (G-CSF), menschlichem Wachstumshormon (hGH), Erythropoietin (EPO), Glukagon, Insulin, Wachstumshormon-Releasing-Hormon, Wachstumshormon-Releasing-Peptid, Interferone, Interferon-Rezeptoren, G-Protein-gekoppelte Rezeptoren, Interleukine, Interleukin-Rezeptoren, Enzyme, Interleukin-bindendes Protein, Zytokin-bindendes Protein, Makrophagen-Aktivierungsfaktor, Makrophagen-Peptid, B-Zell-Faktor, T-Zell-Faktor, Protein A, Allergie-Inhibitor, Zellnekrose-Glykoprotein, Immunotoxin, Lymphotoxin, Tumor-Nekrose-Faktor, Tumorsuppressor, Metastasen-Wachstumsfaktor, α-1-Antitrypsin, Albumin, α-Lactalbumin, Apolipoprotein-E, hochglykosyliertes Erythropoietin, Angiopoietine, Hämoglobin, Thrombin, Thrombinrezeptor-aktivierendes Peptid, Thrombomodulin, Blutfaktoren VII, VIIa, VIII, IX und XIII, Plasminogen-aktivierender Faktor, Fibrin-bindendes Peptid, Urokinase, Streptokinase, Hirudin, Protein C, C-reaktives Protein, Lenin-Inhibitor, Kollagenase-Inhibitor, Superoxiddismutase, Leptin, Thrombozyten-Wachstumsfaktor, Epithel-Wachstumsfaktor, epidermaler Wachstumsfaktor, Angiostatin, Angiotensin, Knochenwachstumsfaktor, Knochen stimulierendes Protein, Calcitonin, Atriopeptin, Knorpel induzierender Faktor, Elcatonin, Bindegewebe aktivierender Faktor, Inhibitor des Gewebefaktor-Signalwegs, Follikel stimulierendes Hormon, luteinisierendes Hormon, luteinisierendes Hormon freisetzendes Hormon, Nervenwachstumsfaktor, Nebenschilddrüsenhormon, Relaxin, Sekretin, Somatomedin, insulinähnlicher Wachstumsfaktor, Nebennierenrindenhormon, Cholecystokinin, Pankreas-Polypeptid, Gastrin-Releasing-Peptid, Corticotropin-Releasing-Faktor, schilddrüsenstimulierendes Hormon, Autotaxin, Lactoferrin, Myostatin, Inkretine, GLP-1/GIP-Dualagonist, gastrisches inhibitorisches Polypeptid (GIP), trigonaler GLP-1/GIP/Glukagon-Agonist, Zelloberflächenantigene, von Viren abgeleitete Impfstoffantigene, monoklonale Antikörper, polyklonale Antikörper und Antikörperfragmente.
  23. Das Verfahren nach Anspruch 22, wobei das physiologisch aktive Polypeptid ein trigonaler GLP-1/GIP/Glukagon-Agonist, Glukagon oder ein Analogon davon ist und/oder eine beliebige Aminosäuresequenz der SEQ ID NOS: 1 bis 6 enthält.
  24. Das Verfahren nach Anspruch 15, wobei die Fc-Region des Immunglobulins von IgG1, IgG2, IgG3 oder IgG4 abgeleitet ist.
  25. Verwendung einer Zusammensetzung zur Herstellung eines lang wirkenden Wirkstoffkonjugats, wobei die Zusammensetzung eine Verbindung mit einer Struktur der nachstehenden Formel 2 oder ein Stereoisomer, ein Solvat oder ein pharmazeutisch akzeptables Salz davon umfasst: wobei der Wirkstoff ein physiologisch aktives Polypeptid ist:
    Figure imgb0025
     wobei in der obigen Formel 2, n von 200 bis 250 ist.
  26. Ein Verfahren zur Herstellung eines lang wirkenden Konjugats eines physiologisch aktiven Polypeptids, wobei das Verfahren umfasst:
    Herstellung eines Konjugats durch Linking einer Verbindung mit einer Struktur der nachstehenden Formel 2 oder eines Stereoisomers, eines Solvats oder eines pharmazeutisch akzeptablen Salzes davon an ein physiologisch aktives Polypeptid:
    Figure imgb0026
    wobei in der obigen Formel 2, n von 200 bis 250 ist.
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JP6937773B2 (ja) 2016-03-07 2021-09-22 ハンミ ファーマシューティカル カンパニー リミテッド ポリエチレングリコール誘導体及びその用途
KR20180091773A (ko) 2017-02-07 2018-08-16 한미약품 주식회사 비펩티드성 중합체 링커 화합물, 그 링커 화합물을 포함하는 결합체, 및 이들의 제조방법
US11357861B2 (en) 2017-09-29 2022-06-14 Hanmi Pharm. Co., Ltd Protein complex comprising non-peptidyl polymer-coupled fatty acid derivative compound as linker and preparation method therefor

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JP2022532445A (ja) 2022-07-14
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CN114945587A (zh) 2022-08-26
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US20230248843A1 (en) 2023-08-10
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US12491258B2 (en) 2025-12-09
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US20220257788A1 (en) 2022-08-18
US11717577B2 (en) 2023-08-08

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